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Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production.

UNLABELLED: There is growing interest in engineering Pseudomonas putida KT2440 as a microbial chassis for the conversion of renewable and waste-based feedstocks, and metabolic engineering of P. putida relies on the understanding of the functional relationships between genes. In this work, independent component analysis (ICA) was applied to a compendium of existing fitness data from randomly barcoded transposon insertion sequencing (RB-TnSeq) of P. putida KT2440 grown in 179 unique experimental conditions. ICA identified 84 independent groups of genes, which we call fModules (functional modules), where gene members displayed shared functional influence in a specific cellular process. This machine learning-based approach both successfully recapitulated previously characterized functional relationships and established hitherto unknown associations between genes. Selected gene members from fModules for hydroxycinnamate metabolism and stress resistance, acetyl coenzyme A assimilation, and nitrogen metabolism were validated with engineered mutants of P. putida. Additionally, functional gene clusters from ICA of RB-TnSeq data sets were compared with regulatory gene clusters from prior ICA of RNAseq data sets to draw connections between gene regulation and function. Because ICA profiles the functional role of several distinct gene networks simultaneously, it can reduce the time required to annotate gene function relative to manual curation of RB-TnSeq data sets. IMPORTANCE: This study demonstrates a rapid, automated approach for elucidating functional modules within complex genetic networks. While Pseudomonas putida randomly barcoded transposon insertion sequencing data were used as a proof of concept, this approach is applicable to any organism with existing functional genomics data sets and may serve as a useful tool for many valuable applications, such as guiding metabolic engineering efforts in other microbes or understanding functional relationships between virulence-associated genes in pathogenic microbes. Furthermore, this work demonstrates that comparison of data obtained from independent component analysis of transcriptomics and gene fitness datasets can elucidate regulatory-functional relationships between genes, which may have utility in a variety of applications, such as metabolic modeling, strain engineering, or identification of antimicrobial drug targets.

Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.

Medium chain‐length polyhydroxyalkanoates (mcl‐PHAs) are efficiently synthesized from fatty acids. It was hypothesized that under non‐axenic conditions, the anti‐microbial properties of fatty acids would reduce growth of microbial competitors and allow Pseudomonas putida LS46 to dominate the population resulting in non‐sterile mcl‐PHA production. Non‐sterile batch and fed‐batch cultures produced similar final mcl‐PHA content, monomer composition, and productivity during 24–27 h cultivations when compared to sterile control cultures for initial octanoic acid concentrations ranging from 5 to 40 mM at an initial pH of 6.5. In the absence of a P. putida inoculum, growth was eventually observed in non‐sterile medium after a lag period of up to 120 h, the length of which was dependent on the octanoic acid concentration. The efficacy of prolonged non‐sterile cultivation was tested using a sequential batch reactor (SBR). After 28 days (672 h), 1.62 g L−1 of total biomass was observed, of which the mcl‐PHA content was 47%. This resulted in a PHA titer of 0.77 g L−1, and a volumetric PHA productivity of 32 mg L−1 h−1. The polymer composition remained stable throughout at 87.0 ± 2.7% C8 monomers, and short‐chain length (scl‐) monomers were not detected. This study is the first of its kind to report that purely mcl‐PHAs were produced after prolonged periods in a non‐sterile environment and demonstrate that medium chain‐length fatty acids exert a strong selective pressure toward organisms that synthesize mcl‐PHA. This suggests an opportunity for mcl‐PHA production in open continuous cultivation, which could reduce both fixed and operating costs. [ABSTRACT FROM AUTHOR]

The continuous emergence of new illegal compounds, particularly psychoactive chemicals, poses significant challenges for current drug detection methods. Developing new protocols and kits for each new drug requires substantial time, effort, and dedicated manpower. Whole-cell bacterial bioreporters have been proven capable of detecting diverse hazardous compounds in both laboratory and field settings, identifying not only single compounds but also chemical families. We present the development of a microbial bioreporter for the detection of cocaine, the nervous system stimulant that is the second-most widely used illegal drug in the US. Escherichia coli was transformed with a plasmid containing a bacterial luxCDABEG bioluminescence gene cassette, activated by a cocaine-responsive signaling cascade. The engineered bioreporter is demonstrated to be a sensitive and specific first-generation detection system for cocaine, with detection thresholds of 17 ± 8 μg/L and 130 ± 50 μg/L in a buffer solution and in urine, respectively. Further improvement of the sensor's performance was achieved by altering the nucleotide sequence of the PBen gene promoter, the construct's sensing element, using accelerated site-directed evolution. The applicability of ready-to-use paper strips with immobilized bioreporter cells was demonstrated for cocaine detection in aqueous solutions. [ABSTRACT FROM AUTHOR]

cis,cis-Muconic acid (MA) is a bio-based chemical that can be converted to direct replacement chemicals or performance-advantaged bioproducts. We recently engineered the bacterium Pseudomonas putida KT2440 for the co-utilization of glucose and xylose to produce MA. This study evaluates the effect of additional genetic modifications, media composition, and bioprocess strategy on MA titer, productivity, and yield in bioreactor cultivations. We achieve a MA titer of 47.2 g L−1, a productivity of 0.49 g L−1 h−1, and a yield of 0.50 C-mol C-mol−1 from glucose and xylose supplemented with 5% (v/v) corn steep liquor with a P. putida strain harboring the deletion of gacS. Additionally, we demonstrate efficient MA production from corn stover-derived sugars and scalability to 150 L bioreactors. Techno-economic analysis and life cycle assessment predict that adipic acid, derived from catalytic hydrogenation of MA, can achieve a selling price as low as $2.60 per kg, approaching cost parity and reducing greenhouse gas emissions by up to 80% relative to fossil carbon-based adipic acid. [ABSTRACT FROM AUTHOR]

As the habitats of bacteria, soil pore network and surface properties control the distribution, adhesion, and motility of bacteria in soils. These physical processes in turn influence bacterial accesses to nutrients and bacterial interactions. Our understanding on the pore- and surface-mediated bacterial interactions is currently limited. In this research, we evaluated the effects of soil pore confinement and surface adhesion on conjugation-based bacterial interactions. The interaction was measured by plasmid transfer between donor and recipient cells within the population of soil bacterium Pseudomonas putida. We found that the presence of porous sand media led to a net increase in conjugation frequency compared to sand-free liquid control. The increase is attributed to the facilitated effect of pore confinement on the collision of bacteria within pores. In contrast, bacterial adhesion to sand surfaces under elevated ionic strength conditions decreased the conjugation frequency as a result of mobility reduction on the surface. Such collision and adhesion mechanisms jointly drive the conjugation as a function of pore and surface properties of porous media. These results provide valuable insights into the roles of soil pores and surfaces in regulating horizontal gene transfer, an essential cell-to-cell interaction sustaining key processes of soil ecology and health. [ABSTRACT FROM AUTHOR]

This study investigated the germination response to temperature of seeds of nine Arabidopsis thaliana ecotypes. They are characterized by a similar temperature dependency of seed germination, and 10 °C and 29 °C were found to be suboptimal low and high temperatures for all nine ecotypes, even though they originated from regions with diverse climates. We tested the potential of four PGPR strains from the genera Pseudomonas and Bacillus to stimulate seed germination in the two ecotypes under these suboptimal conditions. Biopriming of seeds with only the biofilm-forming strain Pseudomonas putida KT2440 significantly increased the germination of Cape Verde Islands (Cvi-0) seeds at 10 °C. However, biopriming did not significantly improve the germination of seeds of the widely utilized ecotype Columbia 0 (Col-0) at any of the two tested temperatures. To functionally investigate the role of KT2440's biofilm formation in the stimulation of seed germination, we used mutants with compromised biofilm-forming abilities. These bacterial mutants had a reduced ability to stimulate the germination of Cvi-0 seeds compared to wild-type KT2440, highlighting the importance of biofilm formation in promoting germination. These findings highlight the potential of PGPR-based biopriming for enhancing seed germination at low temperatures. [ABSTRACT FROM AUTHOR]

Biosensors are used to detect and quantify chemicals produced in industrial microbiology with high specificity, sensitivity, and portability. Most biosensors, however, are limited by the need for transcription factors engineered to recognize specific molecules. In this study, we overcome the limitations typically associated with traditional biosensors by engineering Pseudomonas putida for whole-cell sensing of a variety of chemicals. Our approach integrates fluorescent reporters with synthetic auxotrophies within central metabolism that can be complemented by target analytes in growth-coupled setups. This platform enables the detection of a wide array of structurally diverse chemicals under various conditions, including co-cultures of producer cell factories and sensor strains. We also demonstrate the applicability of this versatile biosensor platform for monitoring complex biochemical processes, including plastic degradation by either purified hydrolytic enzymes or engineered bacteria. This microbial system provides a rapid, sensitive, and readily adaptable tool for monitoring cell factory performance and for environmental analyzes. Biosensors are powerful tools for quantification of a wide range of molecules but require extensive engineering for each analyte. Here, the authors engineered a robust environmental bacterium for sensing a diverse set of chemicals, such as lactate and PET degradation products, via growth-coupling [ABSTRACT FROM AUTHOR]

There is high market demand for (−)‐menthol because of its unique aroma and cooling properties. The first bottleneck encountered in the biosynthesis of (−)‐menthol in Escherichia coli is the low hydroxylation efficiency of (−)‐limonene‐3‐hydroxylase. Few known enzymes can hydroxylate the third carbon atom of (−)‐limonene. In this work, initially, we searched for new enzymes and screened a library of cytochrome P450BM3 variants aiming to improve catalysis of this reaction, but did not identify any variants with improved activity or regioselectivity. Then, through protein engineering of a cytochrome P450 monooxygenase variant from Pseudomonas putida (PpcamY96F/V247L), we obtained variant PpcamF87W/Y96F/L244I/V247L/V396A with regioselectivity of 93% (starting from 70%) toward (−)‐limonene without losing activity. Because of the improvement in regioselectivity, its turnover number was 1.5 times that of the starting enzyme. We analyzed the reasons for the improvement through molecular dynamics simulations. The new variant also exhibited 95% regioselectivity toward (+)‐limonene. This enzyme variant can be applied in biosynthesis of (−)‐menthol. [ABSTRACT FROM AUTHOR]

The present study aimed to characterize the biosurfactants synthesized by lactic acid bacteria (LAB) obtained from fermented foods, optimize the conditions for increasing the yield of biosurfactants and explore their antimicrobial and antibiofilm potential. Out of the 26 LAB isolates, isolate BS2 showed the highest biosurfactant production as indicated in the oil displacement test, drop collapse and emulsification activity. BS2 was identified as Lactiplantibacillus plantarum 1625 using 16S-rRNA gene sequencing and phylogenetic analysis. The biosurfactant produced by BS2 was identified as an anionic glycol-lipo-proteins by employing Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The biosurfactants produced by L. plantarum 1625 demonstrated strong antibacterial and antibiofilm characteristics against pathogenic strains such as Staphylococcus aureus MTCC 1049, Escherichia coli MTCC 1587, and Pseudomonas putida MTCC 1655. The minimal inhibition concentration value of antibacterial activity was found to be 0.1 mg/mL with the inhibition percentage ranging from 90 to 95%. Further, the effect of temperature, pH, and substrate composition on biosurfactant production was also studied to enhance it production using the Box-Behnken Design approach of Response surface methodology (RSM). Application of biosurfactant led to a considerable decrease in biofilm-forming harmful bacteria, as proven by scanning electron microscopy analysis. The results highlight the potential uses of biosurfactants in distinct industries, and biotechnological contexts, especially in the creation of new antimicrobial and antibiofilm agents. [ABSTRACT FROM AUTHOR]

Food contamination has emerged as a significant global health concern, posing substantial challenges to the food industry. Bacteria are the primary cause of foodborne diseases. Consequently, it is crucial to develop accurate and efficient sensing platforms to detect foodborne bacteria in food products. Among various detection methods, biosensors have emerged as a promising solution due to their portability, affordability, simplicity, selectivity, sensitivity, and rapidity. Electrospun nanofibers have gained increasing popularity in enhancing biosensor performance. These nanofibers possess a distinctive three-dimensional structure, providing a large surface area and ease of preparation. This review provides an overview of the electrospinning technique, nanofibers and nanofiber-based biosensors. It also explores their mechanisms and applications in the detection of foodborne bacteria such as Salmonella, Listeria monocytogenes (L. monocytogenes), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas putida (P. putida). [ABSTRACT FROM AUTHOR]

Salinity stress negatively affects the growth and yield of crops worldwide. Onion (Allium cepa L.) is moderately sensitive to salinity. Beneficial microorganisms can potentially confer salinity tolerance. This study investigated the effects of endomycorrhizal fungi (M), Pseudomonas putida (Ps) and their combination (MPs) on onion growth under control (0 ppm), moderate (2000 ppm) and high (4000 ppm) NaCl salinity levels. A pot experiment was conducted with sandy loam soil and onion cultivar Giza 20. Results showed that salinity reduced growth attributes, leaf pigments, biomass and bulb yield while increasing oxidative stress markers. However, individual or combined inoculations significantly increased plant height, bulb diameter and biomass production compared to uninoculated plants under saline conditions. MPs treatment provided the highest stimulation, followed by Pseudomonas and mycorrhizae alone. Overall, dual microbial inoculation showed synergistic interaction, conferring maximum benefits for onion growth, bulbing through integrated physiological and biochemical processes under salinity. Bulb yield showed 3.5, 36 and 83% increase over control at 0, 2000 and 4000 ppm salinity, respectively. In conclusion, combined application of mycorrhizal-Pseudomonas inoculations (MPs) effectively mitigate salinity stress. This approach serves as a promising biotechnology for ensuring sustainable onion productivity under saline conditions. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas putida KT2440 has emerged as a promising host for industrial bioproduction. However, its strictly aerobic nature limits the scope of applications. Remarkably, this microbe exhibits high bioconversion efficiency when cultured in an anoxic bio-electrochemical system (BES), where the anode serves as the terminal electron acceptor instead of oxygen. This environment facilitates the synthesis of commercially attractive chemicals, including 2-ketogluconate (2KG). To better understand this interesting electrogenic phenotype, we studied the BES-cultured strain on a systems level through multi-omics analysis. Inspired by our findings, we constructed novel mutants aimed at improving 2KG production. Results: When incubated on glucose, P. putida KT2440 did not grow but produced significant amounts of 2KG, along with minor amounts of gluconate, acetate, pyruvate, succinate, and lactate. 13C tracer studies demonstrated that these products are partially derived from biomass carbon, involving proteins and lipids. Over time, the cells exhibited global changes on both the transcriptomic and proteomic levels, including the shutdown of translation and cell motility, likely to conserve energy. These adaptations enabled the cells to maintain significant metabolic activity for several weeks. Acetate formation was shown to contribute to energy supply. Mutants deficient in acetate production demonstrated superior 2KG production in terms of titer, yield, and productivity. The ∆aldBI ∆aldBII double deletion mutant performed best, accumulating 2KG at twice the rate of the wild type and with an increased yield (0.96 mol/mol). Conclusions: By integrating transcriptomic, proteomic, and metabolomic analyses, this work provides the first systems biology insight into the electrogenic phenotype of P. putida KT2440. Adaptation to anoxic-electrogenic conditions involved coordinated changes in energy metabolism, enabling cells to sustain metabolic activity for extended periods. The metabolically engineered mutants are promising for enhanced 2KG production under these conditions. The attenuation of acetate synthesis represents the first systems biology-informed metabolic engineering strategy for enhanced 2KG production in P. putida. This non-growth anoxic-electrogenic mode expands our understanding of the interplay between growth, glucose phosphorylation, and glucose oxidation into gluconate and 2KG in P. putida. [ABSTRACT FROM AUTHOR]

Here we report on light-triggered generation of local flow utilizing a bio-compatible non-ionic photo-active surfactant. The mechanism is based on diffusioosmotic phenomenon, where the gradient of relative concentration with respect to different chemical species near a surface leads to an osmotic pressure gradient driving liquid flow along the surface. The application of a photo-responsive surfactant allows for easy and reversible changes in concentration gradient by positioning a light source at the desired place. Along with the so-inscribed concentration gradient one can change reversible the direction and strength of the flow even in a closed system. The phenomenology of light-driven diffusioosmotic flow (LDDO) can be used in a rather flexible way: colloids can be gathered or dispersed and bio-compatibility extends the range of colloid types also to living microorganisms such as soil bacterium Pseudomonas putida. We show that DO flow can be considered a versatile method to set hydrodynamic conditions along the sample for investigating the motility of living cells. Further advantages of employing LDDO are the flexibility of flow generation in a reversible way and with spatiotemporal control, without the need to either change the channel geometry by loading a different device, or the periphery of pumps and connectors. [ABSTRACT FROM AUTHOR]

The study was conducted at the Mycotoxin Laboratory, Department of Plant Protection / College of Agriculture / University of Basrah for the period 2023-2024. A set of experiments were performed to evaluate the effectiveness of Beltanol (BT), Carbendazim (MBC) and Thiophanate- methyl (TM) fungicides in controlling Rhizoctonia solani, which causes eggplant root rot. The genetic matching using BLAST revealed that the bacterial isolates that isolated from non-infected eggplant soils were Aeromonas caviae, Enterobacter cloacae and Pseudomonas putida. The toxicity results of fungicides on R. solani also showed the efficiency of the BT in inhibiting the pathogenic fungus by 100% for the tested concentrations of 2, 4, 8, 10, 15, 20, 100, 250, 300, 350, 400 and 500 mg L-1. Both the MBC and TM fungicides demonstrated variation in their efficiency in controlling the pathogen. The toxicity lines confirmed the values of the median effective concentrations (EC50), as they reached 100 and 489.7 mg L-1 for both MBC and TM, respectively. The 50% inhibitory concentration (I50) of BT was estimated at 7.94 mg L-1 for A. caviae, E. cloacae and P. putida respectively. While it was 12.58 mg L-1 for MBC in A. caviae, E. cloacae and P. putida respectively. While it was 31.62 mg L-1 for TM in the case of A. caviae, E. cloacae and P. putida respectively. [ABSTRACT FROM AUTHOR]

Strawberry cultivation holds significant economic and social promise within Peruvian fruit production. However, conventional management practices have led to the excessive use of agrochemicals in this crop. This study proposes an organic approach to strawberry production, integrating less environmentally harmful technologies. The aim was to assess microbial inoculation by using Trichoderma sp. and Pseudomonas putida and the application of organic amendments on strawberry seedlings of the commercial cultivar "San Andreas". A field experiment was established with evaluations in the vegetative and productive stages. Results indicate that the co-inoculation of Trichoderma sp. and Pseudomonas putida increased leaf area by 7%, and enhanced the aerial part's fresh and dry biomass by 13% and 28%, respectively, compared to treatment without microbial inoculation. Concurrently, compost application increased the leaf number and aerial dry biomass by 22% and 19% at the end of the vegetative stage, respectively, compared to treatment without organic amendment. In addition, it reduced the days for flowering, maintaining the fruit's physicochemical attributes. Regarding yield, the amendments application significantly enhanced fruit weight per plant by 40%, especially when applied together with Trichoderma sp., and co-inoculation increased the number of fruits per meter square by 22%. These findings highlight the potential of technologies such as microbial inoculation and organic amendments to enhance strawberry yields and to gradually reduce the use of synthetic fertilizers. [ABSTRACT FROM AUTHOR]

In recent years, the design–build–test–learn (DBTL) cycle has become a key concept in strain engineering. Modern biofoundries enable automated DBTL cycling using robotic devices. However, both highly automated facilities and semi-automated facilities encounter bottlenecks in clone selection and screening. While fully automated biofoundries can take advantage of expensive commercially available colony pickers, semi-automated facilities have to fall back on affordable alternatives. Therefore, our clone selection method is particularly well-suited for academic settings, requiring only the basic infrastructure of a biofoundry. The automated liquid clone selection (ALCS) method represents a straightforward approach for clone selection. Similar to sophisticated colony-picking robots, the ALCS approach aims to achieve high selectivity. Investigating the time analogue of five generations, the model-based set-up reached a selectivity of 98 ± 0.2% for correctly transformed cells. Moreover, the method is robust to variations in cell numbers at the start of ALCS. Beside Escherichia coli, promising chassis organisms, such as Pseudomonas putida and Corynebacterium glutamicum, were successfully applied. In all cases, ALCS enables the immediate use of the selected strains in follow-up applications. In essence, our ALCS approach provides a 'low-tech' method to be implemented in biofoundry settings without requiring additional devices. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis and associated bacteria are important urogenital pathogens. This study aimed to investigate the effect of T. vaginalis and its associated bacteria on semen and sperm parameters related to male fertility. Urine and semen samples were collected from 97 males, whose ages between 18-50 years. Semen and sperm parameters, such as semen volume, sperm count, sperm concentration, sperm motility, leucocytes/pus cells, and erythrocytes, were evaluated according to the WHO instructions. Conventional-PCR and real-time quantitative PCR were used to detect T. vaginalis infection. Amie's-transport, blood-agar, MacConkey-agar, Chocolate-agar, Thayer-Martin-agar, and Mannitol-salt-agar were used to culture bacteria in urine, and the Vitek2 system was used to differentiate bacteria. Samples that showed positive microscopic examination for bacteria but were not culturable were diagnosed using PCR. T. vaginalis and bacterial infection rates were 31.8% and 17.5%, respectively. T. vaginalis, bacteria, and both together caused a decrease in some qualitative and quantitative parameters of semen and sperm such as a decline in sperm total count by 46.5, 63.46, and 65.67% respectively, sperm concentration by 6.27, 15.37, and 44.27% respectively, sperm motility by 67.63, 66.18 and 66.75% respectively, semen volume by 42.2, 36.4 and 63.86% respectively. They caused increases of 100.92, 16.89, and 84.56%, respectively, and sperm death by 36.58, 80.0%, 93%, and 60.77%, respectively. In addition, they caused an increase in WBCs/pus cells and RBCs in the semen and urine. Four bacteria species, Pantoea spp., Sphingomonas paucimobilis, Staphylococcus aureus, Methylobacterium spp. were detected in urine, and 10 species, Enterobacter aerogenes, Serratia marcescens, Pseudomonas putida, Sphingomonas paucimobilis, Aeromonas sobria, Burkholderia cepacia, Staphylococcus aureus, Aerococcus viridans, Pseudomonas androgens and Staphylococcus epidermidis were detected in semen. In addition, PCR identified three unculturable species: one in urine (Holomonas spp.), and two in semen (Lactobacillus jensenii and Holomonas spp.), S. aureus had the highest frequency in urine and semen of (32%). [ABSTRACT FROM AUTHOR]

Capillary assemblies and microfluidic devices used for bacterial chemotaxis assays have certain inherent limitations. This opens opportunities for innovation in the area. The present study describes an innovative economical device called chemotaxis plate and also a method to use this device for chemotaxis assay. Two type cultures, Pseudomonas putida MCC 2989 and Bacillus subtilis MCC 2049, chemotactic to L-aspartate, were used to validate the new device and establish the protocol for assay. 100 to 1000 fold higher number of cells were recovered in presence of chemoattractant as compared to control (p < 0.05). This novel assay technique showed 100% sensitivity and 99.21% specificity for chemotaxis assay of Pseudomonas putida MCC 2989 towards 3 mM L-aspartate over 50 min assay time. The device was also used to isolate bacteria chemotactic to caffeine directly from environmental samples. Very high chemotaxis response indices were reported for the first-time using chemotaxis plate. [ABSTRACT FROM AUTHOR]

Bacteria indigenous to oil-contaminated water exhibited diverse metabolic capabilities in degrading various aromatic and monoaromatic hydrocarbons. Out of the 28 bacterial strains isolated from the wastewater, each was cultivated with at least one hydrocarbon, including kerosene, naphthalene, toluene, diesel, or aniline. Among these strains, Pseudomonas putida AD-128 emerged as one of the most effective polyaromatic hydrocarbon (PAH) degraders. Following a 6-day treatment period, strain P. putida AD-128 demonstrated proficiency in degrading various PAHs, including naphthalene, phenanthrene, and fluorine. After 6 days of incubation at 20 °C, the degradation of Naphthalene (NAP) notably increased. Gas Chromatography Mass Spectrometry analysis identified the degraded compounds, including pyruvic acid, salicylaldehyde, D-gluconic acid, and catechol. Optimal NAP degradation was observed at 20 °C and pH 6.0, with increased agitation speed correlating with enhanced bacterial growth and heightened degradation, particularly evident after 6 days at 20 °C. Peptone emerged as the most effective among the four nitrogen supplements (ammonium sulfate, potassium nitrate, beef extract, and peptone), significantly reducing residual naphthalene in the medium. The isolated indigenous bacterium, P. putida AD-128, exhibits robust capabilities in degrading PAHs under optimized conditions, making it a valuable asset for environmental management initiatives. [ABSTRACT FROM AUTHOR]

Microbial consortia for disease suppression involve combining multiple beneficial microorganisms to enhance their effectiveness in plant disease management. In present study, development of microbial consortia for the management of major diseases in coconut and citrus was carried out using bacteria Pseudomonas fluorescens, Pseudomonas putida (striata), Bacillus subtilis and fungi - Trichoderma reesei, T. harzianum, T. asperellum against major pathogens viz. Ganoderma lucidum, Thielaviopsis paradoxa, Phytopthora palmivora, Lasiodiplodia theobromae isolated from the coconut rhizosphere, and Fusarium solani isolated from the citrus rhizosphere. The promising fungal and bacterial antagonists were identified and studied for compatibility. Non-volatile compounds of consortia inhibited the test pathogens with an increase in concentration from 10 % to 75% with fungal consortia and bacterial consortia and also with mixed consortia which is composed of bacterial consortia + fungal consortia. Superior growth suppression was recorded with mixed consortia even at 10% concentration (59.44% to 65.83%) against the test pathogens in the ascending order of L. theobromae (59.44%) T. paradoxa (63.89%), G. lucidum (65.83%), P. palmivora (63.61%) and F. solani (62.78%). A similar trend was observed in 75% concentration where inhibition observed in the order of Thielaviopsis paradoxa (90.28%), G. lucidum (89.44%), F. solani (82.50%), L. theobromae (81.94%) and P. palmivora (81.39%). Volatile effect by bacterial consortia recorded the superior inhibition on test pathogens in the order of Ganoderma (85.28%), F. solani (75.28%), T. paradoxa (71.94%), P. palmivora (71.67%) and L. theobrome (67.50%) compared to the individual bioagents. Similarly, the fungal consortia showed the superior inhibitory effect on test pathogens in the order of G. lucidum (83.25%), P. palmivora (82.50%), L. theobromae (83.06%), F. solani (80.56%) and T. paradoxa (73.61%). Since there was no zone of inhibition between the strains, the interactions between Pseudomonas and Bacillus strains of Trichoderma spp. were compatible with one another. Neem cake recorded superior CFU population from 9.43 X 106 CFU at seven days by T. asperellum. Shelf life study on mixed consortia with bacterial + fungal bioagents in talc formulation indicated that all the bacterial and fungal CFU count recorded in 106 dilution for 90 days. [ABSTRACT FROM AUTHOR]

This study aimed to elucidate the potential of Pseudomonas species to degrade furfuryl alcohol (FA), a widely used furanic compound. FA-based resins and biopolymers have applications in various sectors, leading to the gradual discharge of FA into the environment through industrial wastewater. The objective was to find an eco-friendly solution for removing FA residues from industrial effluents, thereby eliminating the hazardous effects of FA. Pseudomonas species have emerged as potential bacterial genera for biodegradation of aromatic compounds; therefore, this study examined the effectiveness of Pseudomonas putida MTCC 1194 and Pseudomonas aeruginosa MTCC 1034 for FA degradation. Significant shifts in the fingerprint regions were observed using Fourier-transform infrared (FTIR) spectroscopy, providing evidence of successful FA degradation. A more than 50% reduction in FA content was observed by both strains, even at high concentrations (500 mgl− 1), as determined by spectrophotometric and high-performance liquid chromatography (HPLC) analysis. Liquid chromatography-mass spectrometry (LC-MS) identified the metabolite as 2-furoic acid (113 m/z). Both bacterial cultures followed substrate inhibition kinetics, and the specific growth rate best fit the Aiba and Haldane models for P. putida and the Haldane and Edwards models for P. aeruginosa. Furthermore, immobilized bacterial cells maintained approximately 50% FA degradation (300 mgl− 1) over five degradation cycles, highlighting their stability and reusability. The phytotoxicity bioassay demonstrated the non-hazardous nature of the metabolites formed after FA biodegradation. These findings underscore the potential of these strains for effective FA bioremediation, paving the way for their use in industrial effluent treatment. [ABSTRACT FROM AUTHOR]

Abstract Salinity stress negatively affects the growth and yield of crops worldwide. Onion (Allium cepa L.) is moderately sensitive to salinity. Beneficial microorganisms can potentially confer salinity tolerance. This study investigated the effects of endomycorrhizal fungi (M), Pseudomonas putida (Ps) and their combination (MPs) on onion growth under control (0 ppm), moderate (2000 ppm) and high (4000 ppm) NaCl salinity levels. A pot experiment was conducted with sandy loam soil and onion cultivar Giza 20. Results showed that salinity reduced growth attributes, leaf pigments, biomass and bulb yield while increasing oxidative stress markers. However, individual or combined inoculations significantly increased plant height, bulb diameter and biomass production compared to uninoculated plants under saline conditions. MPs treatment provided the highest stimulation, followed by Pseudomonas and mycorrhizae alone. Overall, dual microbial inoculation showed synergistic interaction, conferring maximum benefits for onion growth, bulbing through integrated physiological and biochemical processes under salinity. Bulb yield showed 3.5, 36 and 83% increase over control at 0, 2000 and 4000 ppm salinity, respectively. In conclusion, combined application of mycorrhizal-Pseudomonas inoculations (MPs) effectively mitigate salinity stress. This approach serves as a promising biotechnology for ensuring sustainable onion productivity under saline conditions.

The flavoenzyme nicotine oxidoreductase (NicA2) is a promising injectable treatment to aid in the cessation of smoking, a behavior responsible for one in ten deaths worldwide. NicA2 acts by degrading nicotine in the bloodstream before it reaches the brain. Clinical use of NicA2 is limited by its poor catalytic activity in the absence of its natural electron acceptor CycN. Without CycN, NicA2 is instead oxidized slowly by dioxygen (O2), necessitating unfeasibly large doses in a therapeutic setting. Here, we report a genetic selection strategy that directly links CycN-independent activity of NicA2 to growth of Pseudomonas putida S16. This selection enabled us to evolve NicA2 variants with substantial improvement in their rate of oxidation by O2. The encoded mutations cluster around a putative O2 tunnel, increasing flexibility and accessibility to O2 in this region. These mutations further confer desirable clinical properties. A variant form of NicA2 is tenfold more effective than the wild type at degrading nicotine in the bloodstream of rats.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Strawberry cultivation holds significant economic and social promise within Peruvian fruit production. However, conventional management practices have led to the excessive use of agrochemicals in this crop. This study proposes an organic approach to strawberry production, integrating less environmentally harmful technologies. The aim was to assess microbial inoculation by using Trichoderma sp. and Pseudomonas putida and the application of organic amendments on strawberry seedlings of the commercial cultivar “San Andreas”. A field experiment was established with evaluations in the vegetative and productive stages. Results indicate that the co-inoculation of Trichoderma sp. and Pseudomonas putida increased leaf area by 7%, and enhanced the aerial part’s fresh and dry biomass by 13% and 28%, respectively, compared to treatment without microbial inoculation. Concurrently, compost application increased the leaf number and aerial dry biomass by 22% and 19% at the end of the vegetative stage, respectively, compared to treatment without organic amendment. In addition, it reduced the days for flowering, maintaining the fruit’s physicochemical attributes. Regarding yield, the amendments application significantly enhanced fruit weight per plant by 40%, especially when applied together with Trichoderma sp., and co-inoculation increased the number of fruits per meter square by 22%. These findings highlight the potential of technologies such as microbial inoculation and organic amendments to enhance strawberry yields and to gradually reduce the use of synthetic fertilizers.

In this study, the biosynthesis of polyhydroxyalkanoates (PHAs) was carried out using Pseudomonas putida and Pseudomonas aeruginosa. These PHAs were produced using reagent-grade glycerol and crude glycerol as the carbon sources. The objective was to compare the production of PHAs and to functionalize these polymers with silver nanoparticles to provide antibacterial properties for potential biomedical applications. The findings from the physical and chemical analyses confirmed the successful synthesis and extraction of PHAs, achieving comparable yields using both crude glycerol and reagent-grade glycerol as carbon sources across both strains. Approximately 16% higher PHAs production was obtained using Pseudomonas putida compared to Pseudomonas aeruginosa, and no significant difference was observed in the production rate of PHAs between the two carbon sources used, which means that crude glycerol could be utilized even though it has more impurities. Notably, PHAs functionalized with silver nanoparticles showed improved antibacterial effectiveness, especially those derived from reagent-grade glycerol and the Pseudomonas aeruginosa strain. [ABSTRACT FROM AUTHOR]

Aims This study aimed to evaluate the potential of endophytic plant growth-promoting bacterium (PGPB), Pseudomonas putida A32, to mitigate drought stress in two bell pepper genotypes, Amfora 19 and Amfora 26, and to assess the genotype-specific responses to bacterial treatment. Methods and results The isolate P. putida A32 was selected for its remarkable beneficial properties, exhibiting 13 out of 14 traits tested. Under drought conditions, Amfora 26 showed increased relative water content and decreased H2O2 and malondialdehyde following bacterial treatment, while Amfora 19 exhibited enhanced growth parameters but responded less to bacterial treatment regarding drought parameters. However, Amfora 19 displayed inherent drought tolerance mechanisms, as indicated by lower stress parameters compared to Amfora 26. Conclusions The study emphasizes the importance of genotype-specific responses to PGPB treatment and the mechanisms of drought tolerance in peppers. Pseudomonas putida A32 effectively mitigated drought stress in both genotypes, with differential responses influenced by plant genotype. Our study confirmed our initial hypothesis that Amfora 19, as a genotype tolerant to biotic stress, is also more tolerant to abiotic stress. Understanding these interactions is crucial for the development of customized strategies to improve plant productivity and tolerance to drought. [ABSTRACT FROM AUTHOR]

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome but largely lack genetic tools comparable to those available in model species such as Escherichia coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework, along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208, and demonstrate conserved functionality in 11 additional environmental isolates of Rhodococcus, Nocardia, and Gordonia. This toolkit encompasses five Mycobacteriale origins of replication, five broad-host-range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux toward the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species. [ABSTRACT FROM AUTHOR]

Background and Objective: The present study was performed to evaluate the effect of plant growthpromoting rhizobacteria (PGPR), trichoderma fungi and humic acid on the yield and physiological characteristics of wheat (Var. Ofogh). Materials and Methods: This study was carried out as a factorial experiment based on randomized complete block design with three factors and three replications at the research field of the Kashan during 2017-2018 growing season. The first factor was plant growth promoting rhizobacteria such as Azotobacter chroococcum + Azospirillum lipoferum + Pseudomonas putida (application and non-application), the second factor was two concentrations of trichoderma fungi (application and non-application) and three factor was humic acid soil application (0 and 200 kg. ha-1 ). Results: The Results showed that plant growth-promoting bacteria, trichoderma fungus and humic acid led to an increase in plant height, chlorophyll a and b, grain yield and yield components of wheat. Growth-promoting bacteria, trichoderma and humic acid led to an increase of 43.8, 21.7 and 6% in grain yield compared to the control, respectively. The combined use of growth-promoting bacteria, trichoderma fungus and humic acid led to an increase in grain yield, yield components and harvest index compared to single application and control.The highest grain yield was obtained with the combined application of growth-promoting bacteria and trichoderma fungus amounting to 7466.7 kg per hectare and the highest harvest index was obtained with the combined application of biological fertilizers at the rate of 32.3% Conclusion: In general, it is possible to obtain a significant grain yield increase in wheat with the combined application of plant growth-promoting bacteria, trichoderma and humic acid. [ABSTRACT FROM AUTHOR]

The present study was proposed with the idea to screen and isolate efficient low-density polyethylene (LDPE) degrading novel bacterial strains from the plastic-contaminated dumping site. The identification of the bacterial isolate was performed with the help of microbiological and molecular characterization approaches. The screening of the best isolate was performed based on its growth in Bushnell-Hass broth supplemented with LDPE sheets as the sole carbon source. The molecular characterization revealed that the isolate WD4 showed a similarity with the Pseudomonas aeruginosa species. A comparative analysis of Pseudomonas aeruginosa WD4 identified in the current study with Pseudomonas putida MTCC 2445 strain was performed. The present study demonstrated that the bacterial isolate showed 9.2% degradation of LDPE films while Pseudomonas putida revealed a 6.5% weight reduction after 100 days of incubation at 37 °C. The end products of the LDPE degradation were analysed using Fourier transform infrared spectroscopy (FTIR) and gas chromatography-mass spectrometry (GC–MS). The LDPE degradation products eluted include fatty acids such as octadecanoic, hexadecanoic acid, dodecanal, and octyl palmitoleate, alkanes, and some of the unknown compounds after 100 days of microbial treatment with the isolated strain. The detailed analysis of the by-products generated in the current study indicates their contribution to the biochemical pathway of LDPE degradation. The profound scope lies in the scalability of these bacterial strains at the industrial level to combat the LDPE waste and similar plastic garbage problems, globally. [ABSTRACT FROM AUTHOR]

Surfactants are amphiphilic molecules that are capable of mixing water and oil. Biosurfactants are eco-friendly, low-toxicity, and stable to a variety of environmental factors. Optimizing conditions for microorganisms to produce biosurfactants can lead to improved production suitable for scaling up. In this study, we compared heterologous expression levels of the luminescence system luxCDABE operon controlled by regulatable promoters araC-PBAD and its strong version araC-PBAD-SD in Escherichia coli K12, Pseudomonas aeruginosa PAO1, and P. putida KT2440. Real-time monitoring of luminescence levels in the three strains indicated that luxCDABE controlled by araC-PBAD-SD promoter with 0.2% arabinose supplementation in P. putida produced the highest level of luminescence. By using the araC-PBAD-SD promoter-controlled rhlAB expression in P. putida, we were able to produce mono-rhamnolipid at a level of 1.5 g L−1 when 0.02% arabinose was supplemented. With the same system to express olsB, lyso-ornithine lipid was produced at a level of 10 mg L−1 when 0.2% arabinose was supplemented. To our knowledge, this is the first report about optimizing conditions for lyso-ornithine lipid production at a level up to 10 mg L−1. Taken together, our results demonstrate that regulatable araC-PBAD-SD promoter in P. putida KT2440 is a useful system for heterologous production of biosurfactants. [ABSTRACT FROM AUTHOR]

In nature, methylmercury (MeHg) is primarily generated through microbial metabolism, and the ability of bacteria to methylate Hg(II) depends on both bacterial properties and environmental factors. It is widely known that, as a metabolic analog, molybdate can inhibit the sulfate reduction process and affect the growth and methylation of sulfate-reducing bacteria (SRB). However, after it enters the cell, molybdate can be involved in various intracellular metabolic pathways as a molybdenum cofactor; whether fluctuations in its concentration affect the growth and methylation of aerobic mercury methylating strains remains unknown. To address this gap, aerobic γ-Proteobacteria strains Raoultella terrigena TGRB3 (B3) and Pseudomonas putida TGRB4 (B4), as well as an obligate anaerobic δ-Proteobacteria strain of the SRB Desulfomicrobium escambiense CGMCC 1.3481 (DE), were used as experimental strains. The growth and methylation ability of each strain were analyzed under conditions of 500 ng⋅L-1 Hg(II), 0 and 21% of oxygen, and 0, 0.25, 0.50, and 1 mM of MoO4 2-. In addition, in order to explore the metabolic specificity of aerobic strains, transcriptomic data of the facultative mercury-methylated strain B3 were further analyzed in an aerobic mercuric environment. The results indicated that: (a) molybdate significantly inhibited the growth of DE, while B3 and B4 exhibited normal growth. (b) Under anaerobic conditions, in DE, the MeHg content decreased significantly with increasing molybdate concentration, while in B3, MeHg production was unaffected. Furthermore, under aerobic conditions, the MeHg productions of B3 and B4 were not influenced by the molybdate concentration. (c) The transcriptomic analysis showed several genes that were annotated as members of the molybdenum oxidoreductase family of B3 and that exhibited significant differential expression. These findings suggest that the differential expression of molybdenum-binding proteins might be related to their involvement in energy metabolism pathways that utilize nitrate and dimethyl sulfoxide as electron acceptors. Aerobic bacteria, such as B3 and B4, might possess distinct Hg(II) biotransformation pathways from anaerobic SRB, rendering their growth and biomethylation abilities unaffected by molybdate. [ABSTRACT FROM AUTHOR]

Soft corals are marine invertebrates belonging to the class Anthozoa and are rich in bioactive substances that provide their defense and protection from predators, pathogens, and other unfavorable organisms. Seven soft coral species belonging to the genera Sarcophyton and Sinularia were collected from Omani water and extracted and their antibacterial and antifungal properties were tested using the agar-disk diffusion method and a MicroResp technique. Potential compounds that were present in the species were characterized using GC-MS analysis. The results showed that the soft coral species have anti-bacterial activity against Gram-negative bacteria Pseudomonas putida and Pseudomonas tunicata. The GC-MS analysis found the presence of antibacterial, antifungal, antiviral, anticancer, antioxidant agents, agrochemicals, and medicine chemicals. Three novel compounds were found in a Sarcophyton sp. In addition, the compound Andrographolide found in a Sarcophyton sp. has been recently studied as an antiviral agent against SAR-CoV-2. Moreover, the compound Decosane which is found in Sinularia sp. has been used as an anti-parasitic agent against the female Anopheles mosquito to treat malaria in Pakistan. This study highlighted the importance of Omani soft corals as a source of novel bioactive compounds. [ABSTRACT FROM AUTHOR]

The contamination of food by spoilage microorganisms and/or pathogens is a concern in the food industry. Pseudomonas species, Gram-negative, aerobic, and psychrotrophic bacteria, can develop in food products stored at low temperatures. The species causing spoilage in raw meat include Pseudomonas fragi, Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas lundensis. Psychrotrophic pseudomonads cause bad odors, off-tastes, discoloration, and mucus production in raw meat stored under refrigeration and aerobic conditions. Some Pseudomonas species that develop in meats stored at low temperatures are strong biofilm producers. The spoilage activities of pseudomonads are linked to a cell-to-cell communication system known as quorum sensing (QS). The QS system is responsible for encoding genes related to pathogenicity by sensing population density through molecules produced under adverse conditions. Understanding the spoilage mechanisms of pseudomonads in meat is important for inhibiting their development. Research on the QS system of meat pseudomonads is limited. This review discusses the biodiversity, spoilage properties, biofilm formation abilities, and QS effects of Pseudomonas species on meat spoilage. [ABSTRACT FROM AUTHOR]

Pseudomonas putida , a Gram-negative aerobe widely used in biotechnological applications, has attracted attention as a novel exoelectrogen in BESs. BESs exploit the EET capability of exoelectrogens in microbial fuel cells, microbial electrolysis cells, microbial electrosynthesis, and microbial desalination cells. EET enables anaerobic respiration with various external redox-active surfaces such as electrodes and metal oxides, allowing energy conversion and electron transfer both inside and outside the cell. The performance of BESs with P. putida as the exoelectrogen is affected by key factors such as substrates, electrode materials, and EET pathways. BESs with P. putida as the exoelectrogen have been used for removal of organics, the biosynthesis of valuable chemicals, and environmental toxicity testing. Bioelectrochemical systems (BESs), a group of promising integrated systems that combine the advantages of biotechnology and electrochemical techniques, offer new opportunities to address environmental and energy challenges. Exoelectrogens capable of extracellular electron transfer (EET) are the critical factor enabling electrocatalytic activity in BESs. Pseudomonas putida , an aerobe widely used in environmental bioremediation, the biosynthesis of valuable chemicals, and energy bioproduction, has attracted much attention due to its unique application potential in BESs. This review provides a comprehensive understanding of the working principles, key factors, and applications of BESs using P. putida as the exoelectrogen. The challenges and perspectives for the development of BESs with P. putida as the exoelectrogen are also proposed and discussed. [ABSTRACT FROM AUTHOR]

Pseudomonas putida has become an increasingly important chassis for producing valuable bioproducts. This development is not least due to the ever‐improving genetic toolbox, including gene and genome editing techniques. Here, we present a novel, one‐plasmid design of a critical genetic tool, the pEMG/pSW system, guaranteeing one engineering cycle to be finalized in 3 days. The pEMG/pSW system proved in the last decade to be valuable for targeted genome engineering in Pseudomonas, as it enables the deletion of large regions of the genome, the integration of heterologous gene clusters or the targeted generation of point mutations. Here, to expedite genetic engineering, two alternative plasmids were constructed: (1) The sacB gene from Bacillus subtilis was integrated into the I‐SceI expressing plasmid pSW‐2 as a counterselection marker to accelerated plasmid curing; (2) double‐strand break introducing gene I‐sceI and sacB counterselection marker were integrated into the backbone of the original pEMG vector, named pEMG‐RIS. The single plasmid of pEMG‐RIS allows rapid genome editing despite the low transcriptional activity of a single copy of the I‐SceI encoding gene. Here, the usability of the pEMG‐RIS is shown in P. putida KT2440 by integrating an expression cassette including an msfGFP gene in 3 days. In addition, a large fragment of 12.1 kb was also integrated. In summary, we present an updated pEMG/pSW genome editing system that allows efficient and rapid genome editing in P. putida. All plasmids designed in this study will be available via the Addgene platform. [ABSTRACT FROM AUTHOR]

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O -demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O -demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O -demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida , and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway. Enzyme kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O- demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA , involved in formaldehyde detoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms , yielding a platform strain for efficient O -demethylation of lignin-related aromatic compounds to value-added products. • Depolymerization of lignin, followed by bioconversion, is a promising strategy to convert lignin to valuable products. • Methoxylated monomers, like vanillate, must undergo O -demethylation to enter catabolic pathways in aerobic bacteria. • Vanillate O-demethylation pathways were compared in Pseudomonas putida KT2440, focusing on the LigM and VanAB systems. • ALE optimized each pathway, but the optimized VanAB pathway demonstrated faster vanillate utilization. • Mutations were reverse-engineered into P. putida to generate an improved vanillate biocatalyst for valorization of lignin. [ABSTRACT FROM AUTHOR]

Sunscreen has been used for thousands of years to protect skin from ultraviolet radiation. However, the use of modern commercial sunscreen containing oxybenzone, ZnO, and TiO 2 has raised concerns due to their negative effects on human health and the environment. In this study, we aim to establish an efficient microbial platform for production of shinorine, a UV light absorbing compound with anti-aging properties. First, we methodically selected an appropriate host for shinorine production by analyzing central carbon flux distribution data from prior studies alongside predictions from genome-scale metabolic models (GEMs). We enhanced shinorine productivity through CRISPRi-mediated downregulation and utilized shotgun proteomics to pinpoint potential competing pathways. Simultaneously, we improved the shinorine biosynthetic pathway by refining its design, optimizing promoter usage, and altering the strength of ribosome binding sites. Finally, we conducted amino acid feeding experiments under various conditions to identify the key limiting factors in shinorine production. The study combines meta-analysis of 13C-metabolic flux analysis, GEMs, synthetic biology, CRISPRi-mediated gene downregulation, and omics analysis to improve shinorine production, demonstrating the potential of Pseudomonas putida KT2440 as platform for shinorine production. • Meta-Analysis of 13C-MFA and GEMs show P. putida to be an ideal host for shinorine production. • CRISPRi-mediated gene downregulation of PP_1444 improves shinorine production. • Shinorine was produced at high titers and amino acid supplementation improved the titer. • Shinorine accumulates exclusively in the supernatant in P. putida cultures. [ABSTRACT FROM AUTHOR]

Background The aac(6 ′)-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. Objectives To identify sources for the aac(6 ′)-Im fragment found in A. baumannii. Methods MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6 ′)-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. Results Among >60 000 non- Acinetobacter draft genomes in the MRSN collection, the aac(6 ′)-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6 ′)-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6 ′)-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6 ′)-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6 ′)-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6 ′)-Im cassette was different. Conclusions The aac(6 ′)-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype. [ABSTRACT FROM AUTHOR]

The metagenome-assembled genome (MAG) sequences of Pseudomonas putida PP14A and PP20A were obtained by metagenomic sequencing from the gut microbiomes of a female and a male patient both 24 years old from the same household presenting to a health outreach laboratory with complaint of headache, and occasional diarrhoea in Mararaba, Nasarawa State, Nigeria. The phylogenetic relationship observed between the two PP MAGs with other Pseudomonas spp MAGs from human, points to the global spread of Pseudomonas putida through human activity and migration. [ABSTRACT FROM AUTHOR]

To achieve future food security, reliable crop protection will be critically important. Development of biological control for soil borne disease is accepted as a safe, durable and ecofriendly alternative for chemical management strategy. Pseudomonads possess many traits like production antibiotics, lytic enzymes, siderophores, hydrogen cyanide (HCN) that make them well suited as biocontrol and growth-promoting agents. An attempt was taken to isolate and identify different fluorescent Pseudomonads and to evaluate their antagonistic ability. Twelve fluorescent Pseudomonads isolates were collected from different crop rhizosphere from different locations of northern parts of West Bengal. Isolates characterized morphologically and biochemically. All the isolates were gram negative rods with smooth and glossy colony on kings B and nutrient agar medium with convex elevation but variation was found in respect to pigment production. A huge variation was found in respect to biochemical characterization. From the result of biochemical analysis, the isolated sp. can be identified as Pseudomonas fluorescens or Pseudomonas putida or Pseudomonas aeruginosa. Some of the isolate produce green, some isolates produce yellowish green and rest of them produce red/pink pigments. Isolates showed a huge variation in respect to pathogenicity against soil borne pathogen Slerotium rolfsii. Percent inhibition varied from 31 to 74 %. Isolate 3 and 8 can be efficient biocontrol agent as highest growth inhibition of the pathogen Sclerotium rolfsii was obtained by the isolate 8 (74.37%) followed by isolate 3 (63.15%). In respect to growth parameter isolate 8, 9 and 10 were found better among the isolates where as in respect to disease reduction isolate 3, 8, 9 and 11 were promising under in vivo study. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis. [ABSTRACT FROM AUTHOR]

The aim of this research was to estimate the immunopotentiation effect of brown algae Padina boergesenii water extract on Nile tilapia, Oreochromis niloticus through resistance to Pseudomonas putida infection. Gas Chromatography Mass Spectrometry was utilized to characterize the seaweed phytoconstituents. One hundred and twenty-six fish were divided in triplicates into two equal groups corresponding to two diet variants that used to feed Nile tilapia for 20 successive days: a basal (control), and P. boergesenii water extract supplemented group. Fish samples were collected at 10-days intervals throughout the experiment. Serum biochemical constituents, total antioxidant capacity (TAC), and some immune related genes expression of the spleen and intestinal tissues of experimental fish were studied, as well as histological examination of fish immune tissues. Moreover, following 20 days of feeding, the susceptibility of Nile tilapia to P. putida infection was evaluated to assess the protective effect of the used extract. The findings indicated that the studied parameters were significantly increased, and the best immune response profiles were observed in fish fed P. boergesenii water extract for 20 successive days. A bacterial challenge experiment using P. putida resulted in higher survival within the supplemented fish group than the control. Thus, the lowered post-challenge mortality of the fish may be related to the protection provided by the stimulation of the innate immune system, reduced oxidative stress by higher activity of TAC, and elevated levels of expression of iterleukin-1beta (IL-1β), beta-defensin (β-defensin), and natural killer-lysin (NKl). Moreover, the constituents of the extract used showed potential protective activity for histological features of the supplemented fish group when compared to the control. Collectively, this study presents a great insight on the protective role of P. boergesenii water extract as an additive in Nile tilapia feed which suggests its potential for improving the immune response against P. putida infection. [ABSTRACT FROM AUTHOR]

Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species. ATP dynamics influence bioproduction yet are largely unexplored in this context. Here, authors unravel ATP dynamics across various conditions, identify carbon sources which boost ATP levels and bioproduction, and uncover metabolic bottlenecks, shedding light on how ATP dynamics can be used to enhance bioproduction. [ABSTRACT FROM AUTHOR]

Oil-degrading bacteria have the ability to degrade alkanes present in crude oil because of a special enzymatic system, the alkane hydroxylase complex (AlkH). The mechanism for the transport and degradation of alkanes present in crude oil remains unclear, especially related to the first step in hydrocarbons oxidation. In this work, we present a novel model of the crude oil biodegradation mechanism by considering the contact between the oil drop and the cell and calculating the mass transfer coefficients in three oleophilic bacteria (B. licheniformis, P. putida and P. glucanolyticus). The mass transfer coefficients are evaluated under critical time conditions, when the kinetics and mass transport are in balance, and the difference in the values obtained (kL α = 1.60 × 10−3, 5.25 × 10−4 and 6.19 × 10−4 m/d, respectively) shows the higher value of the mass transfer coefficient and higher biodegradation potential for B. licheniformis. Because the morphology of the cells has been analyzed by optical and electron microscopy, in the proposed model, the increase in the size of the cells in P. glucanolyticus compared to P. putida exhibits higher values of the mass transfer coefficients and this is attributed, as a novel statement, to a bigger window for alkanes transport (contact area) when the external area of the cell is bigger. [ABSTRACT FROM AUTHOR]

Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene terephthalate) (PET). Recently, the use of EG as a starting material for the production of bio-fuels or bio-chemicals is gaining attention as a sustainable process since EG can be derived from materials not competing with human food stocks including CO2, syngas, lignocellulolytic biomass, and PET waste. In order to design and construct microbial process for the conversion of EG to value-added chemicals, microbes capable of catabolizing EG such as Escherichia coli, Pseudomonas putida, Rhodococcus jostii, Ideonella sakaiensis, Paracoccus denitrificans, and Acetobacterium woodii are candidates of chassis for the construction of synthetic pathways. In this mini-review, we describe EG catabolic pathways and catabolic enzymes in these microbes, and further review recent advances in microbial conversion of EG to value-added chemicals by means of metabolic engineering. Key points: • Ethylene glycol is a potential next-generation feedstock for sustainable industry. • Microbial conversion of ethylene glycol to value-added chemicals is gaining attention. • Ethylene glycol-utilizing microbes are useful as chassis for synthetic pathways. [ABSTRACT FROM AUTHOR]

Cellular metabolic activity can be detected by tetrazolium-based colorimetric assays, which rely on dehydrogenase enzymes from living cells to reduce tetrazolium compounds into colored formazan products. Although these methods have been used in different fields of microbiology, their application to the detection of bacteria with plastic-degrading activity has not been well documented. Here, we report a microplate-adapted method for the detection of bacteria metabolically active on the commercial polyester polyurethane (PU) Impranil®DLN using the tetrazolium salt 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT). Bacterial cells that are active on PU reduce XTT to a water-soluble orange dye, which can be quantitatively measured using a microplate reader. We used the Pseudomonas putida KT2440 strain as a study model. Its metabolic activity on Impranil detected by our novel method was further verified by Fourier-transform infrared spectroscopy (FTIR) analyses. Measurements of the absorbance of reduced XTT at 470 nm in microplate wells were not affected by the colloidal properties of Impranil or cell density. In summary, we provide here an easy and high-throughput method for screening bacteria active on PU that can be adapted to other plastic substrates. [ABSTRACT FROM AUTHOR]